Within the field of cell culture as applied to bio-pharmaceutical processes there exists a need to separate cells from the media in which they are grown. The desired product from the cell culture may be a molecular species that the cell excretes into the media, a molecular species that remains within the cell, or it may be the cell itself. At production scale, the initial stages of cell culture process typically take place in bioreactors, which may be operated in either batch or continuous mode. Intermediate variations such as repeat batch are practiced as well. In all cases the product must eventually be separated from other process components prior to final purification and product formulation. Cell harvest is a general term applied to these cell separations. Clarification is a term denoting separations wherein a cell-free supernatant (or centrate) is the objective. Cell recovery is a term often applied to separations wherein a cell concentrate is the objective. The present invention is directed to cell harvest separations in large-scale cell culture systems.
Methods currently in common use for cell harvest separations include batch, intermittent, continuous and semi-continuous centrifugation, tangential flow filtration (TFF) and depth filtration. Historically, centrifuges for cell harvest of large volumes of cell culture at production scale are complex multiple use systems that require clean-in-place (CIP) and steam-in-place (SIP) technology to provide an aseptic environment to prevent contamination by microorganisms. At lab scale and for continuous cell harvest processes, smaller systems are currently in use. These smaller lab scale systems are based on pre-sterilized, single-use fluid path components.
Pre-sterilized, single-use components are already used for media storage, mix tanks, hold tanks, and bioreactors, e.g. up to about 2000 liter capacity which are used in the initial stages of the cell culture process, but until recently there were no production scale centrifuges available that used pre-sterilized components for the harvest of such large cell culture batches. The UniFuge centrifuge system, manufactured by Pneumatic Scale Corporation, described in published application US 2010/0167388, the entire disclosure of which is incorporated herein by reference, overcomes such limitations, and successfully processes culture batches in the range of 3-30 liters/minute in quantities of up to about 2000 liters using intermittent processing. Intermittent processing requires periodically stopping both rotation of the centrifuge bowl and the feed flow in order to discharge concentrate. This works well with lower concentration, high viability cultures, in which large batches can be processed, and the cell concentrate discharged relatively quickly and completely.
The current industry trend is toward harvesting highly concentrated and/or low viability cell cultures, which contain a high concentration of cells and cell debris in the feed, sometimes referred to as “high turbidity feeds.” Such high turbidity feeds slow down the processing rate using current technology, because:    1. a slower feed flow rate is required to provide increased residence time in the centrifuge in order to separate small cell debris particles, and    2. the higher concentrations of both cells and cell debris result in the bowl filling rapidly with cell concentrate, which require the bowl to be stopped frequently to discharge concentrate.
These combined factors result in a greatly reduced net throughput rate, and unacceptably long cell harvest processing times. In addition to the increased costs which are associated with a longer processing time, increased time in the centrifuge may also result in a higher degree of product contamination and loss when harvesting low viability cell cultures.
This high concentration of cell and cell debris also results in a cell concentrate with a very high viscosity, which makes it more difficult to completely discharge the cell concentrate, even with a prolonged discharge cycle. In some cases, an additional buffer rinse cycle must be added to obtain a sufficiently complete discharge of concentrate. The need to make either or both of these adjustments to the discharge cycle further increases the processing time, making the challenges of processing a large volume of cell culture more complex and costly.
Scaling up the existing technology, by increasing the bowl size to increase the length of the feeding portion of the intermittent processing cycle is not practical because it would also result in a proportionately longer discharge cycle for the cell concentrate. Another limitation that precludes simple geometric scale-up is variation in scaling of the pertinent fluid dynamic factors. The maximum processing rate of any centrifuge depends on the settling velocity of the particles being separated. The settling velocity is given by a modification of Stokes' law defined by Equation 1:
                    v        =                              Δ            ⁢                                                  ⁢                          ρ              ·              r              ·                              d                2                            ·                              ω                2                                                          18            ·            μ                                              Equation        ⁢                                  ⁢        1            
where v=settling velocity, Δρ is solid-liquid density difference, d is particle diameter, r is radial position of the particle, ω is angular velocity, and μ is liquid viscosity. With respect to scale-up geometry, the radius of the bowl affects the maximum radial position r that particles can occupy. Therefore, if the other parameters in Equation 1 are held constant, an increase in bowl radius leads to an increase in average settling velocity and a gain in throughput for a given separation efficiency. However, as the radius increases it becomes more difficult to maintain the angular velocity of the bowl because of the material strength that would be required, and other engineering limitations. If a decrease in angular velocity is larger than the square root of the proportional increase in radius, then the average settling velocity and the gain in throughput (which is proportional to radius) both decline.
One of the engineering limitations that must be considered is that the angular velocity needed to rotate the larger bowl is not be practical to achieve because of the massive and costly centrifuge drive platform that would be needed.
In addition if the angular velocity is held constant as the radius increases, the forces urging the cells toward the walls of the centrifuge also increase. When the bowl is rotated at sufficiently high angular velocity to create the desired processing efficiency the walls of the container and the cells which accumulate there experience added stress. As to the cells, this can cause cell damage by packing the cells to excessively high concentrations. Cell damage is a drawback in applications wherein cell viability needs to be maintained and can lead to contamination of products that are present in solution in the centrate. The higher viscosity resulting from excessively high cell concentrations is a drawback for complete discharge of the cell concentrate.
Accordingly, there is a need for a centrifuge system, with single use components, which can be used to efficiently separate production scale quantities of cell cultures having a high concentration of cells and cell debris.